FIELD OF THE INVENTION

The present invention relates to crystalline compositions of mammalian 3', 5'-Cyclic Nucleotide Phosphodiesterase 9A (PDE9A), methods of preparing the compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of the composition, methods of identifying ligands of PDE9A using structure based drug design, the use of the three-dimensional crystal structure to design, modify and assess the activity of potential inhibitors, and to the use of such inhibitors for treating a variety of diseases, particularly diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome and/or cardiovascular disease. BACKGROUND OF THE INVENTION Cyclic nucleotide second messengers (cAMP and cGMP) play a central role in signal transduction and regulation of physiologic responses. Their intracellular levels are controlled by the complex superfamily of cyclic nucleotide phosphodiesterase (PDE) enzymes. The PDE superfamily is comprised of metallophosphohydrolases (e.g., Mg2+, and Zn2+) that specifically cleave the 3',5'-cyclic phosphate moiety of cAMP and/or cGMP to produce the corresponding 5'-nucleotide. The sensitivity of physiological processes to cAMP/cGMP signals requires that their levels be precisely maintained within a relatively narrow range in order to provide for optimal responsiveness in a cell. Cyclic nucleotide PDEs provide the major pathway for eliminating the cyclic nucleotide signal for the cell. PDEs are critical determinants for modulation of cellular levels of cAMP and/or cGMP by many stimuli.

Members of the PDE superfamily differ in their tissue distributions, physicochemical properties, substrate and inhibitor specificities and regulatory mechanisms. Based on differences in primary structure of known PDEs, they have been subdivided into two major classes, class I and class II. To date, no mammalian PDE has been included in class II. Class I contains the largest number of PDEs and includes all known mammalian PDEs. Each class I PDE contains a conserved segment of -250-300 amino acids in the carboxyl- terminal portion of the proteins, and this segment has been demonstrated to include the catalytic site of these enzymes. All known class I PDEs are contained within cells and vary in subcellular distribution, with some being primarily associated with the particulate fraction of the cytoplasmic fraction of the cell, others being evenly distributed in both compartments. PDEs from mammalian tissues have been subdivided into 11 families that are derived from separate gene families. The families are named PDE1 , PDE2, PDE3...to PDE11. Within each family, there may be isoenzymes such as PDE1A, PDE1 B and PDE1C, and PDE10A1 and PDE10A2. PDEs within a given family may differ significantly but the members of each family are functionally related to each other through similarities in amino acid sequences, specificities and affinities for cGMP (PDE5, PDE6, and PDE9) or cAMP (PDE4, PDE7, and PDE8) or accommodation of both (PDE1 , PDE2, PDE3, PDE10, and PDE11), inhibitor specificities, and regulatory mechanisms.

Comparison of the amino acid sequences of PDEs suggests that all PDEs may be chimeric multidomain proteins possessing distinct domains that provide for catalysis and a number of regulatory functions. The amino acid sequences of all mammalian PDEs identified to date include a highly conserved region of approximately 270 amino acids located in the carboxy terminal half of the proteins. (Charbonneau, et al., Proc. Natl. Acad., Sci. (USA) 83:9308-9312 (1986)). The conserved domain includes the catalytic site for cAMP and/or cGMP hydrolysis and two putative metal (presumably zinc) binding sites as well as family specific determinants. (Beavo, Physiol. Rev. 75: 725-748 (1995); Francis, et al., J. Biol. Chem. 269:22477-22480 (1994)). The amino terminal region of the various PDEs are highly variable and include other family specific determinants such as : (i) calmodulin binding sites (PDE1 ); (ii) non-catalytic cGMP binding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4); (iv) hydrophobic membrane association sites (PDE3); and (v) phosphorylation sites for either the calmoduline-dependent kinase (II) (PDE1), the cAMP-dependent kinase (PDE1 , PDE3, PDE4), or the cGMP dependent kinase (PDE5) (Beavo, Physiol. Rev. 75:725-748 (1995); Manganiello, et al., Arch. Biochem. Acta 322: 1- 13 (1995); Conti, et al., Physiol. Rev. 75:723-748 (1995); WO 99/42596).

While all known mammalian PDEs are either dimeric or oligomeric, the functional importance of this quaternary structure is not known, and experiments further indicate that in some PDEs the components required for catalyzing hydrolysis of the phosphodiester bond in cAMP or cGMP are contained in a single catalytic domain and that the interactions between two catalytic domains within a dimer or between the catalytic domain and the regulatory domain are not required for this process. (See Francis, S. H. et al., Prog. Nuc. Acid Res Molec. Biol., 65: 1-52, 2001 ).

Human PDE9A represented the first human member of the PDE9 family that had been cloned and characterized. (Fisher, et al., Journal of Biological Chemistry, 273:5:15559-15564 (1998)). By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). (Fisher, et al., (1998)). Of the 22 amino acids conserved among all eight other PDE families, 21 are also conserved in PDE9A. The one change is a very conservative Tyr to Phe change at amino acid 24 of the catalytic domain. All the features noted above are also conserved in the murine and rattus norvegicus PDE9A sequence, which share about 90% amino acid sequence identity with the human sequence in the catalytic domain. (Fisher, et al., (1998)).

Selective inhibition of PDE9A has been investigated for the treatment of various conditions including diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease. A preferred condition comprises diabetes, metabolic syndrome, and/or cardiovascular disease. Several methods have been used in the past and continue to be used to discover selective inhibitors of biomolecular targets such as PDE9. The various approaches include ligand-directed drug discovery (LDD), quantitative structure activity relationship (QSAR) analyses; and comparative molecular field analysis (CoMFA). CoMFA is a particular type of QSAR method that uses statistical correlation techniques for the analysis of the quantitative relationship between the biological activity of a set of compounds with a specified alignment, and their three-dimensional electronic and steric properties. Other properties such as hydrophobicity and hydrogen bonding can also be incorporated into the analysis.

An invaluable component of these drug discovery approaches is structure based design, which is a design strategy for new chemical entities, or optimization of lead compounds identified by other methods using the three-dimensional (3D) structure of the biological macromolecule target obtained by for example, X-ray or nuclear magnetic resonance NMR studies, or from homology models. Analyzing 3-D structures of proteins provides crucial insights into the behavior and mechanics of drug binding and biological activity. Coupled with computational techniques including modeling and simulation, the study of biomolecular interactions provides details of events that may be difficult to investigate experimentally in the laboratory, and can help reveal topological features important for determining molecular recognition. As those skilled in the art will recognize, this information can, in turn, be used for predicting ligand-receptor complex formation, and for designing ligands and protein mutations that produce desired ligand receptor interactions.

Regulation of PDEs is important for controlling myriad physiological functions, including the visual response, smooth muscle relaxation, platelet aggregation, fluid homeostasis, immune responses, and cardiac contractility. PDEs are critically involved in feedback control of cellular cAMP and cGMP levels. The PDE superfamiiy continues to be a major target for pharmacological intervention in a number of medically important maladies including cardiovascular diseases, asthma, depression, and male impotence. For example, PDE5, found in varying concentrations in a number of tissues, has been recognized in recent years as an important therapeutic agent. (See U.K. Patent Application 0126417.5, filed November 2, 2001 ). To that end the quest for specific and potent PDE inhibitors for use in physiological studies and therapeutic settings continues. Thus, obtaining, three- dimensional (3D) structures of PDEs, such as PDE9A, obtained by for example, X-ray or nuclear magnetic resonance NMR studies, or from homology models, and analyzing the structures using computational methods facilitates such discovery efforts.

SUMMARY OF THE INVENTION

The present invention provides crystalline compositions of PDE, and specifically of the catalytic region of PDE9A. The invention further provides methods of preparing said compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of said crystalline compositions, methods of using the atomic structure coordinates in conjunction with computational methods to identify binding site(s), methods to elucidate the three-dimensional structure of homologues of PDE9A, and methods to identify ligands which interact with the binding site(s) to agonize or antagonize the biological activity of PDE9A, methods for identifying inhibitors of PDE9A, pharmaceutical compositions of inhibitors, and methods of treatment of a variety of conditions including, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease using said pharmaceutical compositions.

In a preferred embodiment the invention provides crystalline compositions of the catalytic region of PDE9A.

One aspect of the present invention provides methods for crystallizing a PDE9A polypeptide ligand complex comprising a polypeptide. Preferably the methods for crystallizing a PDE9A polypeptide ligand complex comprising an amino acid sequence spanning the amino acids 239 to 562 listed in SEQ ID NO:1 , or a homologue, an analogue or a variant thereof comprising the steps of: comprising the steps: (a) preparing a mixture of an aqueous solution comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, or a variant thereof, with a ligand in a 2:1 molar ration; (b) mixing said aqueous solution with a reservoir solution comprising a precipitant to from a mixed volume; and (c) crystallizing said mixed volume. Crystallization can be carried out by various techniques known by those skilled in the art, such as for example, batch crystallization, liquid bridge crystallization, or dialysis crystallization. Preferably, the crystallization is achieved using vapor diffusion techniques. An embodiment of the present invention provides crystalline compositions of PDE9A comprising a crystalline form of a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, an analogue or a variant thereof, wherein the crystalline composition has a space group I422 and unit cell dimensions a=b=223.665 A, c= 119.341 A. In a second aspect, the present invention provides vectors useful in methods for preparing a substantially purified C-terminal catalytic domain of PDE9A comprising the polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, an analogue or a variant thereof.

In a third aspect, the present invention provides methods for determining the X-ray atomic structure coordinates of the crystalline compositions at a 2.7 A resolution.

In a fourth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal has substantially similar atomic structure coordinates to the atomic structure coordinates listed in FIG.4 or portions thereof, or any scalable variations thereof. In a fifth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1. A further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 90% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1. Yet another embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 95% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , and which has the atomic structure coordinates listed in FIG. 4. A further embodiment of the present invention provides a crystal comprising an amino acid sequence that is at least 98% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , and which has the atomic structure coordinates listed in FIG. 4 In a sixth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide, or a portion thereof, with atomic structure coordinates having a root mean square deviation from the protein backbone atoms (N, Ca, C, and O) listed in FIG.1 of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A. In a seventh aspect, the present invention provides a scalable, or translatable, three dimensional configuration of points derived from structural coordinates of at least a portion of a PDE9A molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1. In an embodiment of this aspect, the invention also comprises the structural coordinates of at least a portion of a molecule or ε molecular complex that is structurally homologous to a PDΞ9A molecule or molecular complex. On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A from the structural coordinates provided in FIG. 4. In an eight aspect, the present invention provides a computer for producing a three- dimensional representation of:

a. a molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, an analogue, or a variant thereof;

b. a molecule or molecular complex, wherein the atoms of the molecule or molecular complex are represented by atomic structure coordinates that are substantially similar to, or are subsets of the atomic structure coordinates listed in FIG. 4;

c. a molecule or molecular complex, wherein the molecule or molecular complex comprises atomic structure coordinates having a root mean square deviation of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or even 2.5 A from the atomic structure coordinates for the carbon backbone atoms listed in FIG.1 ; or

d. a molecule or molecular complex, wherein the molecule or molecular complex comprises a binding pocket or site defined by the structure coordinates that are substantially similar to the atomic structure coordinates listed in FIG. 4, or a subset thereof, or more preferably the structural coordinates in FIG. 4 corresponding to one or more PDE9A amino acids, conservative replacements, or functional equivalence thereof, in SEQ ID NO: 1 selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516, wherein said computer comprises: (i) a computer-readable data storage medium comprising a data storage medium encoded with computer-readable data, wherein said data comprises the structure coordinates of FIG. 4, or portions thereof, or substantially similar coordinates thereof; (ii) a working memory for storing instructions for processing said computer- readable data; (iii) a central-processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer-machine readable data into said three-dimensional representation; and (iv) a display coupled to said central-processing unit for displaying said representation.

The computer configured according to this aspect of the invention can be used to design and identify potential modulators of PDE9A by, for example commercially available molecular modeling software in conjunction with structure-based drug design as provided herein. In a further embodiment of the present invention the potential modulators designed and identified using the computer configurations according to the present invention can be for example ligands or inhibitors. In yet an even further embodiment of the present invention provides methods for designing a compound that binds to PDE9A using the molecular or molecular complex, comprising selecting a compound by performing structure- based drug design with the atomic structure coordinates determined for the crystal, wherein said selecting is performed in conjunction with computer modeling.

In a ninth aspect, the present invention provides methods involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure. In one embodiment, the method includes crystallizing the molecule or molecular complex, generating an X-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in FIG. 4 to the X-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.

In another embodiment, the present invention provides methods for generating three-dimensional atomic structure coordinates of a protein homologue or a variant of PDE9A using the X-ray coordinates of PDE9A described in FIG. 4, comprising,

a. identifying one or more homologous polypeptide sequences to PDE9A;

b. aligning said sequences with the sequence of PDE9A which comprises a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 ;

c. identifying structurally conserved and structurally variable regions between said homologous sequence(s) and PDE9A;

d. generating three-dimensional three-dimensional coordinates for structurally conserved residues of the said homologous sequence(s) from those of PDE9A using the coordinates listed in FIG. 4;

e. generating conformations for the loops in the structurally variable regions of said homologous sequence(s);

f. building the side-chain conformations for said homologous sequence(s); and

g. combining the three-dimensional coordinates of the conserved residues, loops and side-chain conformations to generate full or partial three-dimensional coordinates for said homologous sequences.

Embodiments of the ninth aspect provide methods, which further comprise refining and evaluating the full or partial three-dimensional coordinates. Thsse methods may thus be used to generate three-dimensional structures for proteins for which heretofore three- dimensional atomic structure coordinates have not been determined. Depending on the extent of sequence homology, the newly generated structure can help to elucidate enzymatic mechanisms, or be used in conjunction with other molecular modeling techniques in structure based drug design.

In the tenth aspect, the present invention provides methods for identifying modulators, such as for example inhibitors, ligands, and the like of PDE9A by providing the coordinates of a molecule of PDE9A to a computerized modeling system; identifying chemical entities that are likely to bind to or interfere with the molecule (e.g., by screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived for bioactivity. Other embodiments the present invention relate to methods for identifying potential modulators for PDE9A or homologues, an analogue or variants thereof comprising:

a. displaying the three dimensional structure of PDE9A enzyme or homologue or variant thereof, or portions thereof, as defined by atomic structure coordinates that are substantially similar to the atomic structure coordinates listed in FIG. 4 on a computer display screen;

b. optionally replacing one or more the enzyme amino acid residues listed in SEQ ID NO:1 , or preferably one or more amino acid residues, conservative replacements, or functional equivalence thereof, selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516, in said three-dimensional structure with a different naturally occurring amino acid or an unnatural amino acid to display a variant structure;

c. optionally conducting ab intio, molecular mechanics or molecular dynamics calculations on the displayed three dimensional structure to generate a modified structure;

d. employing said three-dimensional structure, variant structure, or modified structure to design or select said ligand;

d. synthesizing or obtaining said ligand;

e. contacting said ligand with said enzyme in the presence of one or more substrates; and

f. measuring the ability of said ligand to modulate the activity of said enzyme.

Those skilled in the art can appreciate that the information obtained by the methods for identifying modulators, such as for example inhibitors, ligands and the like of PDE9A, as described above, can be used to iteratively refine or modify the structure of original modulator. Thus, once a modulator is found to modulate the activity of said enzyms, the structural aspects of the modulator may be modified to generate a structural analog of the modulator. This analog can then be used in the above method to identify binding modulators. One of ordinary skill in the art will know the various ways a structure may be modified. In embodiments, preferred modulators are ligands and include selective inhibitors of PDE9A.

In embodiments, the methods further comprise computationally modifying the structure of the ligand; computationally determining the fit of the modified ligand using the three-dimensional coordinates described in FIG. 4, or portions thereof; contacting said modified ligand with said enzyme, or homologue, or variant thereof in an in vitro or in vivo setting; and measuring the ability of said ligand to modulate the activity of said enzyme.

In an eleventh aspect, the present invention provides compositions and pharmaceutical preparations comprising the modulator designed according to any of the above methods. In one embodiment, a composition is provided that includes an inhibitor or ligand designed or identified by any of the above methods. In another embodiment, the composition is a pharmaceutical composition.

In a twelfth aspect, the present invention provides methods for treating conditions, diseases, or symptoms selected from the group consisting of type 1 diabetes, type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and cardiovascular disease, comprising administering to a patient in need of such treatment the pharmaceutical composition of ligands identified by structure-based drug design using the atomic structure coordinates substantially similar to, or portions of, the coordinated listed in FIG. 4.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an orthogonal view of the embodiment of PDE9A in ribbon representation. The compound of Formula 1 is shown in ball-and-stick representation.

Figure 2 is another orthogonal view of the embodiment of the compound of Formula 1 with PDE9A.

Figure 3 is schematic diagram showing the interactions of the compound of Formula 1 with PDE9A.

Figure 4 is a list of the X-ray coordinates of the PDE9A C-terminal catalytic domain crystal as described in the Examples. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to crystalline compositions of PDE9A, three- dimensional X-ray atomic structure coordinates of said crystalline composition, methods of preparing said compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of said crystalline compositions, and methods of using said atomic structure coordinates in conjunction with computational methods to identify binding site(s), or identify ligands which interact with said binding site(s) to agonize or antagonize PDE9A.

For convenience, certain terms employed in the detailed description, examples, and appendant claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "affinity" as used herein refers to the tendency of a molecule to associate with another. The affinity of a drug is its ability to bind to its biological target (receptor, enzyme, transport system, etc.) For pharmacological receptors, affinity can be thought of as the frequency with which the drug, when brought into the proximity of a receptor by diffusion, will reside at a position of minimum free energy within the force field of that receptor.

The term "agonist" as used herein refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.)

The term "analog" as used herein refers to a drug or chemical compound whose structure is related in some way to that of another drug or chemical compound, but whose chemical and biological properties may be quite different.

The term "antagonist" as used herein refers to a drug or a compound that opposes the physiological effects of another. At the receptor level, it is a chemical entity that opposes the receptor-associated responses normally induced by another bioactive agent.

"Atomic coordinates" or "atomic structural coordinates" are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex. Structural coordinates may be obtained using x-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling. Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex. The structural coordinates of the present invention may be modified from the original sets provided in FIG. 4 by mathematical manipulation, such as by inversion or integer additions or subtractions. As such, it is recognized that the structural coordinates of the present invention are relative, and are in no way specifically limited by the actual x, y, z coordinates of FIG. 4. As used herein the term "binding site" refers to a specific region (or atom) in a molecular entity that is capable of entering into a stabilizing interaction with another molecular entity. In certain embodiments the term also refers to the reactive parts of a macromolecule that directly participate in its specific combination with another molecule. In other embodiments, a binding site may be comprised or defined by the three dimensional arrangement of one or more amino acid residues within a folded polypeptide. In further embodiments, the binding site further comprises prosthetic groups, water molecules or metal ions which may interact with one or more amino acid residues. Prosthetic groups, water molecules, or metal ions may be apparent from X-ray crystallographic data, or may be added to an apo protein or enzyme using in silico methods.

The term "bioactivity" refers to PDE9A activity that exhibits a biological property conventionally associated with a PDE9A agonist or antagonist, such as a property that would allow treatment of one or more of the various diseases of the central nervous system.

The term "catalytic domain" as used herein, refers to the catalytic domain of the PDE9A class of enzymes, which features a conserved segment of amino acids in the carboxy-terminal portion of the proteins, wherein this segment has been demonstrated to include the catalytic site of these enzymes. This conserved catalytic domain extends approximately from residue Asp239 to Glu562 of the full length enzyme.

"To clone" as used herein, means obtaining exact copies of a given polynucleotide molecule using recombinant DNA technology. Furthermore, "to clone into" may be meant as inserting a given first polynucleotide sequence into a second polynucleotide sequence, preferably such that a functional unit combining the functions of the first and the second polynucleotides results. For example, without limitation, a polynucleotide from which a fusion protein may be translationally provided, which fusion protein comprises amino acid sequences encoded by the first and the second polynucleotide sequences. Specifics of molecular cloning can be found in a number of commonly used laboratory protocol books such as Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989).

The term "co-crystallization" as used herein is taken to mean crystallization of a preformed protein/ligand complex.

The term "complex" or "co-complex" are used interchangeably and refer to a PDE9A molecule, or a variant, or homologue of PDE9A in covalent or non-covalent association with a substrate, or ligand. The term "contacting" as used herein applies to in silico, in vitro, or in vivo experiments.

The term "functional equivalence" refers to the assembly of one or more amino acid residues that form a binding site in an enzyme. These residues may have one or more intervening residues which are distant from the binding site, and therefore may minimally interact with a ligand in the binding sites. In such occurrences, the binding site may be defined for the purpose of structure based drug design as comprising only a handful of amino acid residues. For example in the case of PDE9A, the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 of SEQ ID No:1. In a preferred embodiment, the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID No:1. Thus any molecular assembly that has a root mean square deviation from the atomic structure coordinates of the protein backbone atoms (N, Ca, C, and O), or the side chain atoms, of one or more of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 of SEQ ID NO:1 , any conservative substitutions thereof, or any functional equivalence of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed will be considered substantially similar to the coordinates listed in FIG.4. The functional equivalents may be different from the peptides described herein at one, two, three, four, or more amino acid positions. In the most common instances, such differences will involve conservative amino acid substitutions. As used herein, the terms "gene", "recombinant gene" and "gene construct" refer to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.

The term "high affinity" as used herein means strong binding affinity between molecules with a dissociation constant K0 of no greater than 1 μM. In a preferred case, the KD is less than 100 nM, 10 nM, 1 nM, 100 pM, or even 10 pM or less. In a most preferred embodiment, the two molecules can be covalently linked (KD is essentially 0).

The term "homologue" as used herein means a protein, polypeptide, oliogpeptide, or portion thereof, having preferably at least 90% amino acid sequence identity with PDE9A enzyme as described in SEQ ID No:1 or SEQ ID No:2 or any catalytic domain described herein, or any functional or structural domain of lipid binding protein. SEQ ID No:1 is the full- length amino acid sequence of the wild-type human PDE9A. SEQ ID No:2 is the amino acid sequence of the wild type carboxy-terminal catalytic domain of human PDE9A that was crystallized in the Examples. While SEQ ID No:3 is the wild-type mus musculus (mouse) PDE9A amino acid sequence, (Soderling, et al., J. Biol. Chem., 273: 15553-15558, 1998) and SEQ ID No:4 is the wild-type rattus norvegicus (rat) PDE9A amino acid sequence, (Andreeua, S.G., Rosenberg, P.A., "Characterization of PDE9A in the rat brain", Submitted (Apr. 2001) to the EMBL/GenBank/PDBJ database), which are both at least 90% identical with the PDE9A enzyme as described in SEQ ID No:1 , can also be used for crystallization and for the design and identification of potential modulators of PDE9A Those of skill in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. As used herein, and for the purpose of this invention, the term "substantially similar atomic structure coordinates" or atomic structure coordinates that are "substantially similar" refers to any set of structure coordinates of PDE9A or PDE9A homologues, or PDE9A variants, polypeptide fragments, described by atomic coordinates that have a root mean square deviation for the atomic structure coordinates of protein backbone atoms (N, Ca, C, and O) of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed- using backbone atoms- of structure coordinates listed in FIG. 4. For the purpose of this invention, structures that have substantially similar coordinates as those listed in FIG. 4 shall be considered identical to the coordinates listed in FIG. 4. The term "substantially similar" also applies to an assembly of amino acid residues that may or may not form a contiguous polypeptide chain, but whose three dimensional arrangement of atomic structure coordinates have a root mean square deviation for the atomic structure coordinates of protein backbone atoms (N, Ca, C, and O), or the side chain atoms, of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed-using backbone atoms, or the side chain atoms- of the atomic structure coordinates of similar or the same amino acids from the coordinates listed in FIG. 4. To clarify further, but not intending to be limiting, an example of an assembly of amino acids may be the amino acid residues that form a binding site in an enzyme. These residues may have one or more intervening residues which are distant from the binding site, and therefore may minimally interact with a ligand in the binding sites. In such occurrences, the binding site may be defined for the purpose of structure based drug design as comprising only a handful of amino acid residues. For example in the case of PDE9A, the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 of SEQ ID No:1. In a preferred embodiment, the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID No:1. Thus any molecular assembly that has a root mean square deviation from the atomic structure coordinates of the protein backbone atoms (N, Ca, C, and O), or the side chain atoms, of one or more of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501, Ala512, Gln513 and Phe516 of SEQ ID NO:1 , any conservative substitutions thereof, or any functional equivalance of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed will be considered substantially similar to the coordinates listed in FIG.4. Those skilled in the art understand that "substantially similar" atomic structure coordinates are considered identical to the coordinates, or portions thereof, listed in FIG. 4. Those skilled in the art further understand that the coordinates listed in FIG. 4 or portions thereof may be transformed into a different set of coordinates using various mathematical algorithms without departing from the present invention. For example, the coordinates listed in Fig. 4, or portions thereof, may be transformed by algorithms which translate or rotate the atomic structure coordinates. Alternatively, molecular mechanics, molecular dynamics or ab intio algorithms may modify the atomic structure coordinates. Atomic coordinates generated from the coordinates listed in FIG. 4, or portions thereof, using any of the aforementioned algorithms shall be considered identical to the coordinates listed in FIG. 4.

The term "in silico" as used herein refers to experiments carried out using computer simulations. In certain embodiments, the in silico methods are molecular modeling methods wherein 3-dimeπsional models of macromolecules or ligands are generated. In other embodiments, the in silico methods comprise computationally assessing ligand binding interactions.

The term "ligand" describes any molecule, e.g., protein, peptide, peptidomimetics, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., which is designed or developed with reference to the crystal structure of PDE9A as represented by the atomic structure coordinates listed in FIG. 4. In one aspect the ligand is an agonist, whereby the molecule upregulates (i.e., activate or stimulate, e.g., by agonizing or potentiating) activity, while in another aspect of the invention the ligand is an inhibitor or antagonist, whereby the molecule down-regulates (i.e., inhibit or suppress, e.g. by antagonizing, decreasing or inhibiting) the activity.

The term "modulate" as used herein refers to both upregulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down-regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting) of an activity.

The term "modulator" as used herein refers to any molecule, e.g., protein, peptide, peptidomimetics, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., which can either upregulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down-regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting) of an activity.

The term "pharmacophore" as used herein refers to the ensemble of steric and electronic features of a particular structure that is necessary to ensure lhs optimal supramolecular interactions with a specific biological target structure and to trigger (or to block) its biological response. A pharmacophore may or may not represent a real molecule or a real association of functional groups. In certain embodiments, a pharmacophore is an abstract concept that accounts for the common molecular interaction capacities of a group of compounds towards their target structure. In certain embodiments, the term can be considered as the largest common denominator shared by a set of active molecules. Pharmacophoric descriptors are used to define a pharmacophore, including H- bonding, hydrophobic and electrostatic interaction sites, defined by atoms, ring centers and virtual points. Accordingly, in the context of enzyme ligands, such as for example agonists or antagonists, a pharmacophore may represent an ensemble of steric and electronic factors which are necessary to insure supramolecular interactions with a specific biological target structure. As such, a pharmacophore may represent a template of chemical properties for an active site of a protein/enzyme - representing these properties' spatial relationship to one another - that theoretically defines a ligand that would bind to that site.

The term "precipitant" as used herein is includes any substance that, when added to a solution, causes a precipitate to form or crystals to grow. Examples of precipitants within the scope of this invention include, but are not limited to, alkali (e.g., Li, Na, or K), or alkaline earth metal (e.g., Mg, or Ca) salts, and transition (e.g., Mn, or Zn) metal salts. Common counterions to the metal ions include, but are not limited to, halides, phosphates, citrates and sulfates.

The term "prodrug" as used herein refers to drugs that, once administered, are chemically modified by metabolic processes in order to become pharmaceutically active. In certain embodiments the term also refers to any compound that undergoes biotransformation before exhibiting its pharmacological effects. Prodrugs can thus be viewed as drugs containing specialized non- toxic protective groups used in a transient manner to alter or to eliminate properties, usually undesireable, in the parent molecule.

The term "receptor" as used here in refers to a protein or a protein complex in or on a cell that specifically recognizes and binds to a compound acting as a molecular messenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.). In a broader sense, the term receptor is used interchangeably with any specific (as opposed to non-specific, such as binding to plasma proteins) drug binding site, also including nucleic acids such as DNA.

The term "recombinant protein" refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the polypeptide encoded by said DNA. This polypeptide may bs one thst is naturally expressed by the host cell, or it may be heterologous to the host cell, or the host cell may have been engineered to have lost the capability to express the polypeptide which is otherwise expressed in wild type forms of the host cell. The polypeptide may also be, for example, a fusion polypeptide. Moreover, the phrase "derived from", with respect to a recombinant gene, is meant to include within the meaning of "recombinant protein" those proteins having an amino acid sequence of a native polypeptide, or an amino acid sequence similar thereto which is generated by mutations, including substitutions, deletions and truncation, of a naturally occurring form of the polypeptide.

As used herein, the term "selective PDE9A inhibitor" refers to a substance, for example an organic molecule that effectively inhibits an enzyme from the PDE9A family to a greater extent than any other PDE enzyme, particularly any enzyme from the PDE 1-9 families or any PDE10 or PDE11 enzyme. In one embodiment, a selective PDE9A inhibitor is a substance, for example, a small organic molecule having a K; for inhibition of PDE9A that is less than about one-half, one-fifth, or one-tenth the Kj that the substance has for inhibition of any other PDE enzyme. In other words, the substance inhibits PDE9A activity to the same degree at a concentration of about one-half, one-fifth, one-tenth or less than the concentration required for any other PDE enzyme. In general a substance is considered to effectively inhibit PDE9A if it has an IC50 or Ki of less than or about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or even 10 nM.

As used herein the term "small molecules" refers to preferred drugs as they are orally available (unlike proteins which must be administered by injection or topically). Size of small molecules is generally under 1000 Daltons, but many estimates seem to range between 300 to 700 Daltons.

By "therapeutically effective" amount is meant that amount which is capable of at least partially reversing the symptoms of the disease. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on a consideration of the species of the mammal, the size of the mammal, the type of delivery system used, and the type of administration relative to the progression of the disease. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. "Transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.

The term "variants" in relation to the polypeptide sequence in SEQ ID NO:1 or SEQ ID NO:2 include any substitution of, variation of, modification of, replacement of, deletion of, or addition or one or more amino acids from or to the sequence providing a resultant polypeptide sequence for an enzyme having PDE9A activity. Preferably the variant, homologue, fragment or portion, of SEQ ID NO:1 or SEQ ID NO:2, comprise a polypeptide sequence of at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, preferably at least 15 contiguous amino acids, preferably at least 20 contiguous amino acids, preferably at least 25 contiguous amino acids, or preferably at least 30 contiguous amino acids.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

The following amino acid abbreviations are used throughout this disclosure:

A = Ala = Alanine T = Thr = Threonine V = VaI = Valine C = Cys = Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = He = lsoleucine N = Asn = Asparagine P = Pro = Proline Q = GIn = Glutamine F = Phe = Phenylalanin D = Asp = Aspartic Acid W = Trp = Tryptophan E = GIu = Glutamic Acid M = Met = Methionine K = Lys = Lysine G = GIy = Glycine R = Arg = Arginine S = Ser = Serine H = His = Histidine

A. Clones and Expressions The nucleotide sequence coding for a PDE9A polypeptide, or functional fragment, including the C-terminal peptide fragment of the catalytic domain of PDE9A protein, derivatives or analogs thereof, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The elements mentioned above are termed herein a "promoter." Thus, the nucleic acid encoding a PDE9A polypeptide of the invention or a functional fragment comprising the C-terminal peptide fragment of the catalytic domain of PDE9A protein, derivatives or analogs thereof, is operationally associated with a promoter in an expression vector of the invention. In preferred embodiments, the expression vector contains the nucleotide sequence coding for the polypeptide comprising the amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1. Both cDNA and genomic sequences can be cloned and expressed under the control of such regulatory sequences. An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can be provided on a recombinant expression vector. As detailed below, all genetic manipulations described for the PDE9A gene in this section, may also be employed for genes encoding a functional fragment, including the C-terminal peptide fragment of the catalytic domain of the PDE9A protein, derivatives or analogs thereof, including a chimeric protein thereof.

Suitable host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

A recombinant PDE9A protein of the invention may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression. (See Sambrook et al., 1989, infra, the pertinent disclosure of which is incorporated by reference herein in its entirety).

A suitable cell for purposes of this invention is one into which the recombinant vector comprising the nucleic acid encoding PDE9A protein is cultured in an appropriate cell culture medium under conditions that provide for expression of PDE9A protein by the cell.

Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). As those skilled in the art will appreciate, expression of PDE9A protein may be controlled by any promoter/enhancer element known in the art, and these regulatory elements must be functional in the host selected for expression.

Vectors containing a nucleic acid encoding a PDE9A protein of the invention can be identified for example, by four general approaches: (1 ) PCR amplification of the desired plasmid DNA or specific mRNA, (2) nucleic acid hybridization, (3) presence or absence of selection marker gene functions, and (4) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g.,. beta.-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding PDE9A protein is inserted within the "selection marker" gene sequence of the vector, recombinant vectors containing the PDE9A protein insert can be identified by the absence of the PDE9A protein gene function. In the fourth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant vector, provided that the expressed protein assumes a functionally active conformation.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention as known by those of skill in the art.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

Vectors can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 19S2, J. Biol. Cham. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311 , filed Mar. 15, 1990).

B. Crystal and Space Groups X-ray structure coordinates define a unique configuration of points in space. Those skilled in the art understand that a set of structure coordinates for a protein or a protein/ligand complex, or a portion thereof, define a relative set of points that, in turn, define a configuration in three dimensions. A similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between atomic structure coordinates remain essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.

One aspect of the present invention relates to a crystalline composition comprising preferably a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1.

In one embodiment, the present invention discloses a crystalline PDE9A molecule comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 complexed with one or more ligands. In another embodiment, the crystallized complex is characterized by the structural coordinates listed in FIG. 4, or portions thereof. In certain embodiments, the atoms of the ligand are within about 4, 7, or 10 angstroms of one or more PDE9A amino acids in SEQ ID NO: 1 preferably selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516, or a conservative replacements, or functional equivalence thereof.

One embodiment of the crystallized complex is characterized as belonging to the I442 space group and has cell dimensions of a=223.665, b=223.665, c=119.341 A, α=β=γ=90.0°. This embodiment is encompassed by the structural coordinates of FIG. 4. The ligand may be a small molecule which binds to a PDE9A catalytic domain defined by SEQ ID NO: 2, or portions thereof, with a K1 of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM, or even 10 nM.

In a certain embodiment, the ligand is the compound of Formula I, [2-(3-lsopropyl-7- oxo-6, 7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid. In certain embodiments, the ligand is a substrate or substrate analog of PDE9A. In certain embodiments, the ligand(s) may be a competitive or uncompetitive inhibitor of PDE9A. In certain embodiments, the ligand is a covalent inhibitor of PDE9A.

Various computational methods can be used to determine whether a molecule or a binding pocket portion thereof is "structurally equivalent," defined in terms of its three- dimensional structure, to all or part of PDE9A or its binding pocket(s). Such methods may be carried out in current software applications, such as the molecular similarity application of QUANTA (Accelrys Inc., San Diego, Calif.). The molecular similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in molecular similarity to compare structures is divided into four steps: (1 ) load the structures to be compared; (2) optionally define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within molecular similarity applications is defined by user input, for the purpose of this invention equivalent atoms are defined as protein backbone atoms (N, Ca, C, and O) for all conserved residues between the two structures being compared. A conserved residue is defined as a residue that is structurally or functionally equivalent (See Table 4 infra). In certain embodiments rigid fitting operations are considered. In other embodiments, flexible fitting operations may be considered.

When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the molecular similarity application.

For the purpose of this invention, any molecule or molecular complex or binding pocket thereof, or any portion thereof, that has a root mean square deviation of conserved residue backbone atoms (N, Ca, C, and O) of less than about 2.5 A, 2.0 A, 1.5 A, 1.0 A, 0.7 A, 0.5 A or even 0.2 A, when superimposed on the relevant backbone atoms described by the reference structure coordinates listed in FIG. 4, is considered "structurally equivalent" to the reference molecule. That is to say, the crystal structures of those portions of the two molecules are substantially identical, within acceptable error. Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structural coordinates listed in FIG. 4, plus or minus a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 2.5 A. More preferably, the root mean square deviation is less than about 1.0 A.

The term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of PDE9A or a binding pocket portion thereof, as defined by the structural coordinates of PDE9A described herein.

The refined x-ray coordinates of the catalytic domain of PDE9A (amino acids 239 to 562 as listed in SEQ ID NO:2), complexed with the compound of Formula 1 ([2-(3-lsopropyl- 7-0X0-6, 7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid), Zn2+, Mg2+, and 49 water molecules are as listed in FIG. 4.

Two orthogonal views of the molecule are shown in FIG. 1 and FIG. 2 and details of the interactions of the inhibitor with protein are shown in FIG. 3.

The structure is composed of a single domain of fourteen α helices and two 310 helices arranged in a compact fold (FIG. 1). The numbering of the helix is shown below. We have the followed the numbering conversion established by Xu et al., Science, 288:1822-25 (2000), and the start and end points of the helices are determined according to Kabsch and Sander, Biopolymers, 22(12): 2577-637 (1983).

α helices Residue range 310 helices Residue range

H1 249-258 A1 464-468

H2 267-288

H3 292-305

H4 313-331

H5 339-351

H6 360-367

H7 370-375

H8 379-395

H9 405-422

H10 425-439

H11 446-463

H12 470-493

H13 509-519

H14 521-531

H15 537-557 Two metal ions are in the catalytic site. The first is determined to be Zn2+, by analogy with PDE4b, and from an analysis of its coordination geometry. The metal is coordinated by His352 (Nε2-Zn 2.2A), His316 (Nε2-Zn 2.2A), Asp462 (Oδ1-Zn 2.0A), and Asp353 (Oδ2-Zn 2.θA). These residues are completely conserved across the PDE gene family. The second metal ion is coordinated to Asp353 (Oδ1-Mg 2.1A) and to a water network that stabilizes the metal environment. Due to the coordination geometry and the relative observed electron density, this second metal ion has been refined as a Mg2+ in accordance with a similar observation in the PDE4 structure. (Xu et al., Science, 288:1822-25 (2000)). One molecule of the inhibitor, ([2-(3-lsopropyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3- d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid) is seen bound within the active site. The inhibitor binding site is bounded by H12 and H13 on one side, and by the N-terminus of H12 and the 310 helix, A1. Protein-inhibitor interactions are shown schematically in the FIG. 3. The majority of the interactions between the inhibitor and the protein are hydrophobic in nature; with only two hydrogen bonds observed (FIG. 2). One hydrogen bond is between Nε2 of the completely conserved Gln513 and the carbonyl oxygens 04 (3.2A) and the second hydrogen bond is between Oε1 of Gln513 and N2(2.5A). The quinazoline ring of the inhibitor appears to make a strong π-stacking interaction with Phe516 on one side and a hydrophobic interaction with Leu480 on the other side. The acetic acid makes no direct interactions with the protein. Accordingly, the present invention provides a molecule or molecular complex that includes at least a portion of a PDE9A and/or substrate binding pocket. In one embodiment, the PDE9A binding pocket includes the amino acids listed in Table 1 , preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, the binding pocket being defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A, from points representing the backbone atoms of the amino acids in Tables 1-3. In another embodiment, the PDE9A substrate binding pocket includes the amino acids selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 from SEQ ID NO:1 , the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516. In a preferred embodiment, the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516. Table 1 : Residues near the binding pocket in PDE9A catalytic domain. Identified residues are 10 A away from the compound of Formula 1

Table 2: Residues near the binding pocket in PDE9A catalytic domain. Identified residues are 7 A away from the compound of Formula 1

Table 3: Residues near the binding pocket in PDE9A catalytic domain. Identified residues are 5 A away from the compound of Formula 1

C. Isolated Polypeptides and Variants

One embodiment of the invention describes an isolated polypeptide consisting of a portion of PDE9A which functions as the binding site when folded in the proper 3-D orientation. One embodiment is an isolated polypeptide comprising a portion of PDE9A, wherein the portion starts at about amino acid residue Asp239, and ends at about amino acid residue Glu562 as described in SEQ ID NO:1 , or a sequence that is at least 95%, or 98% homologous to a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 as listed in SEQ ID NO:1 , such as, for example the polypeptide of the wild-type mus musculus (mouse) PDE9A enzyme, disclosed in SEQ ID No:3, or the wild- type rattus norvegicus (rat) PDE9A.

Another embodiment of the invention comprises crystalline compositions comprising variants of PDE9A. Variants of the present invention may have an amino acid sequence that is different by one or more amino acid substitutions to the sequence disclosed in SEQ ID NO:1 or SED ID NO:2. Embodiments which comprise amino acid deletions and/or additions are also contemplated. The variant may have conservative changes (amino acid similarity), wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine. Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without adversely affecting biological or proposed pharmacological activity may be reasonably inferred in view of this disclosure, and may further be found using computer programs well known in the art, for example, DNAStar® software.

Amino acid substitutions may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as a biological and/or pharmacological activity of the native molecule is retained.

Negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids, with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine; amino acids with aliphatic head groups include glycine, alanine; asparagine, glutamine, serine; and amino acids with aromatic side chains include threonine, phenylalanine, and tyrosine.

Examples of conservative substitutions are set forth in Table 4 as follows:

Table 4:

"Homology" is a measure of the identity of nucleotide sequences or amino acid sequences. In order to characterize the homology, subject sequences are aligned so that the highest percentage homology (match) is obtained, after introducing gaps, if necessary, to achieve maximum percent homology. N- or C-terminal extensions shall not be construed as affecting homology. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. Computer program methods to determine identity between two sequences, for example, include DNAStar® software (DNAStar Inc. Madison, Wl); the GCG® program package (Devereux, J., et al. Nucleic Acids Research (1984) 12(1): 387); BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec Biol (1990) 215: 403). Homology (identity) as defined herein is determined conventionally using the well-known computer program, BESTFIT® (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl, 53711). When using BESTFIT® or any other sequence alignment program (such as the Clustal algorithm from MegAlign software (DNAStar®)) to determine whether a particular sequence is, for example, about 90% homologous to a reference sequence, according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence or amino acid sequence and that gaps in homology of up to about 90% of the total number of nucleotides in the reference sequence are allowed.

Ninety percent of homology is therefore determined, for example, using the BESTFIT® program with parameters set such that the percentage of identity is calculated over the full length of the reference sequence, e.g., SEQ ID NO:1 , and wherein up to 10% of the amino acids in the reference sequence may be substituted with another amino acid. Percent homologies are likewise determined, for example, to identify preferred species, within the scope of the claims appended hereto, which reside within the range of about 90% to 100% homology to SEQ ID NO: 1 as well as the binding site thereof. As noted above, N- or C-terminal extensions shall not be construed as affecting homology. Thus, when comparing two sequences, the reference sequence is generally the shorter of the two sequences. This means that, for example, if a sequence of 50 nucleotides in length with precise identity to a 50 nucleotide region within a 100 nucleotide polynucleotide is compared, there is 100% homology as opposed to only 50% homology.

Although the natural polypeptide of SEQ ID NO: 1 and a variant polypeptide may only possess a certain percentage identity, e.g., 90%, they are actually likely to possess a higher degree of similarity, depending on the number of dissimilar codons that are conservative changes. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or function of the protein. Similarity between two sequences includes direct matches as well a conserved amino acid substitutes which possess similar structural or chemical properties, e.g., similar charge as described in Table 4.

Percentage similarity (conservative substitutions) between two polypeptides may also be scored by comparing the amino acid sequences of the two polypeptides by using programs well known in the art, including the BESTFIT program, by employing default settings for determining similarity.

A further embodiment of the invention is a crystal comprising the coordinates of FIG.4, wherein the amino acid sequence is represented by SEQ ID NO:1. A further embodiment of the invention is a crystal comprising the coordinates of FIG. 4, wherein the amino acid sequence is at least 90%, 95%, or 98% homologous to the amino acid sequence represented by SEQ ID NO:1.

Various methods for obtaining atomic structure coordinates of structurally homologous molecules and molecular complexes using homology modeling are disclosed in US Patent 6,356,845, which is hereby incorporated by reference in its entirety.

D. Structure Based Drug Design

Once the three-dimensional structure of a crystal comprising a PDE9A protein, a functional domain thereof, homologue or variant thereof, is determined, a potential ligand (antagonist or agonist) may be examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (See for example, Morris et al., J. Computational Chemistry, 19:1639-1662 (1998)). This procedure can include in silico fitting of potential ligands to the PDE9A crystal structure to ascertain how well the shape and the chemical structure of the potential ligand will complement or interfere with the catalytic domain of PDE9A. (Bugg et al., Scientific American, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995)).-Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the ligand to the binding site. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential drug will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interfere with the properties of other proteins. This will minimize potential side-effects due to unwanted interactions with other proteins.

One embodiment of the present invention relates to a method of identifying an agent that binds to a binding site on PDE9A catalytic domain wherein the binding site comprises amino acid residues Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID NO: 1 comprising contacting PDE9A with a test ligand under conditions suitable for binding of the test ligand to the binding site, and determining whether the test ligand binds in the binding site, wherein if binding occurs, the test ligand is an agent that binds in the binding site. In certain embodiments, the testing may be carried out in silico using a variety of molecular modeling software algorithms including, but not limited to, DOCK, ALADDIN, CHARMM simulations, AFFINITY, C2-LIGAND FIT, Catalyst, LUDI, CAVEAT, and CONCORD. (Brooks, et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comp.Chem 1983, 4:187-217; E. C. Meng, B. K. Shoichet & I. D. Kuntz. Automated docking with grid-based energy evaluation. J Comp Chem 1992, 13:505-524.

In another embodiment, a potential ligand may be obtained by screening a random oepfda library produced by ε recombinant bacteriophage for example, (Scott εnd Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. ScL, 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)) or a chemical library, or the like. A ligand selected in this manner can be then be systematically modified by computer modeling programs until one or more promising potential ligands are identified. Such analysis has been shown to be effective in the development of HIV protease inhibitors. (Lam et al., Science 263:380-384 (1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design 1 :23-48 (1993); Erickson, Perspectives in Drug Discovery and Design 1 :109-128 (1993)).

Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, of which any one might lead to a useful drug. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized are actually synthesized. Thus, through the use of the three-dimensional structure disclosed herein and computer modeling, a large number of these compounds can be rapidly screened on a computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.

Once a potential ligand (agonist or antagonist) is identified, it can be either selected from a library of chemicals as are commercially available from most large chemical companies or alternatively the potential ligand may be synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design. The potential ligand can be placed into any standard binding assay as well known to those skilled in the art to test its effect on PDE9A activity.

When a suitable drug is identified, a supplemental crystal can be grown comprising a protein-ligand complex formed between a PDE9A protein and the drug. Preferably the crystal effectively diffracts X-rays allowing the determination of the atomic structure coordinates of the protein-ligand complex to a resolution of less than 5.0 Angstroms, more preferably less than 3.0 Angstroms, and even more preferably less than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal can be determined by Molecular Replacement Analysis. Molecular replacement involves using a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a new crystal form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal. Computer programs that can be used include: X-PLOR and AMORE (J. Navaza, Acta Crystallographies ASO, 157-163 (1994)). Once the position and orientation are known, an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences, and the search model is modified to conform to the new structure. Using this approach, it is possible to use the claimed structure of PDE9A to solve the three- dimensional structures of any such PDE9A complexed with a new ligand. Other computer programs that can be used to solve the structures of such STAT crystals include QUANTA; CHARMM; INSIGHT; SYBYL; MACROMODEL; and ICM.

Various in silico methods for screening, designing or selecting ligands are disclosed in US Patent 6,356,845, the pertinent disclosure of which is incorporated by reference herein.

E. Ligands

In one aspect, the present invention discloses ligands which interact with a binding site of the PDE9A catalytic domain defined by a set of points having a root mean square deviation of less than about 2.5 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG.4. A further embodiment of the present invention comprises binding agents which interact with a binding site of PDE9A defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.7, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG. 4. Such embodiments represent variants of the PDE9A crystal.

In another aspect, the present invention describes ligands which bind to a correctly folded polypeptide comprising an amino acid sequence spanning amino acids 239 to 562 listed in SEQ ID NO:1 , or a homologue or variant thereof. In certain embodiments, the ligand is a competitive or uncompetitive inhibitor of PDE9A. In certain embodiments the ligand inhibits PDE9A with an IC50 or Ki of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or 10 nM. In certain embodiments, the ligand inhibits PDE9A with a K, that is less than about one-half, one-fifth, or one-tenth the K1 that the substance has for inhibition of any other PDE enzyme. In other words, the substance inhibits PDE9A activity to the same degree at a concentration of about one-half, one-fifth, one-tenth or less than the concentration required for any other PDE enzyme.

One embodiment of the present invention relates to ligands, such as proteins, peptides, peptidomimetics, small organic molecules, etc., designed or developed with reference to the crystal structure of PDE9A as represented by the coordinates presented herein in FIG. 4, and portions thereof. Such binding agents interact with the binding site of the PDE9A represented by one or more amino acid residues selected from Met425, Glu466, Leu480, Lsu481 , Tyr484, Pha501 , Ala512 Gln513 and Pha51δ. F. Machine Readable Storage Media

Transformation of the structure coordinates for all or a portion of PDE9A, or the PDE9A/ligand complex or one of its binding pockets, for structurally homologous molecules as defined below, or for the structural equivalents of any of these molecules or molecular complexes as defined above, into three-dimensional graphical representations of the molecule or complex can be conveniently achieved through the use of commercially- available software.

The invention thus further provides a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three- dimensional representation of any of the molecule or molecular complexes of this invention that have been described above. In a preferred embodiment, the machine-readable data storage medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex comprising all or any parts of a PDE9A C-terminal catalytic domain or binding pocket, as defined above. In another preferred embodiment, the machine-readable data storage medium is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex defined by the structure coordinates of the amino acids listed in FIG. 4, plus or minus a root mean square deviation from the backbone atoms of said amino acids of not more than 2.5 A.

In an alternative embodiment, the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in FIG. 4, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data.

For example, a system for reading a data storage medium may include a computer comprising a central processing unit ("CPU"), a working memory which may be, e.g., RAM (random access memory) or "core" memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube ("CRT") displays, light emitting diode ("LED") displays, liquid crystal displays ("LCDs"), electroluminescent displays, vacuum fluorescent displays, field emission displays ("FEDs"), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, touch screens, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus. The system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.). The system may also include additional computer controlled devices such as consumer electronics and appliances.

Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.

Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices. By way of example, the output hardware may include a display device for displaying a graphical representation of a binding pocket of this invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. References to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof. Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device. It should be understood that these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data. G. Pharmaceutical Compositions

The present invention contemplates methods for treating certain diseases in a mammal, preferably a human, in need of such treatment using the ligands, and preferably the inhibitors, as described herein. The ligand can be advantageously formulated into pharmaceutical compositions comprising a therapeutically effective amount of the ligand, a pharmaceutically acceptable carrier and other compatible ingredients, such as adjuvants, Freund's complete or incomplete adjuvant, suitable for formulating such pharmaceutical compositions as is known to those skilled in the art. Pharmaceutical compositions containing the ligand can be used for the treatment of a variety of conditions including diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease. A preferred condition comprises diabetes, metabolic syndrome, and/or cardiovascular disease. The pharmaceutical composition is administered to the mammal in a therapeutically effective amount such that treatment of the disease occurs. The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference in their entireties.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology and recombinant DNA, X-ray crystallography, and molecular modeling which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and Il (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuIMs et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Crystallography Made Clear: A Guide For Users Of Macromolecular Models (Gales Rhodes, 2nd Ed. San Diego: Academic Press, 2000). EXAMPLES

Example 1 : Construction and expression of PDE9A wild type catalytic domain

Amino acids 239-562 of human wild type PDE9A (SEQ ID NO:1 ) construct was generated by PCR amplification. The resulting construct was subcloned into a into Ncol/BamHI into pFastBac-1 (Invitrogen, Fredrick, MD) in order to generate recombinant baculovirus using the Bac-to-Bac system (Gibco Carlsbad, CA), corresponding to the amino acids in SEQ ID NO:2. The protein was expressed in SF9. The insect cells were infected with the recombinant baculovirus at a MOI (multiplicity of infection) of 0.5 and harvested 72 hrs. post infection. Pellets of infected cells were frozen at -8O0C for transfer to purification.

Example 2: Purification of PDE9A wild type catalytic domain

Baculovirus cell paste (80 g) containing the over expressed PDE9A N3C2 recombinant protein was resuspended in 3 volumes buffer A, containing 2OmM Tris (trimethoxylmethaneamine) pH 8.0, 15OmM NaCI (sodium chloride), 5% (v/v) glycerol, 2mM TCEP (Tri(2-carboxyethyl) phosphine hydrochloride) and 1 Complete™ protease inhibitor cocktail tablet/50mL (EDTA free) (Roche, Applied Science, Indianapolis, IN). The cells were sonicated using handheld sonicator (VirTis Virsonic 100) for 3 x 30 sonications (level 7), the cell debris was removed by centrifugation at 4°C for 45 minutes at 35,000xg. The resulting clarified supernatant is collected and batch-bound to Ni-NTA Superflow resin (Qiagen) for 30 min at 40C on rocker table. Ni-NTA resin is first washed with dH2O and equilibrated in Ni-NTA buffer A (20 mM Tris 8.0, 150 mM NaCI, 2 mM TCEP, 5% glycerol, 20 mM Imadizole). Batch- bound resin is then loaded into Pharmacia XK-16 column and washed with Ni-NTA Buffer A until baseline is reached. Protein is step eluted with Ni-NTA Buffer B (20 mM Tris 8.0, 150 mM NaCI, 2 mM TCEP, 5% glycerol, 250 mM Imadizole). Entire elutant peak is collected and dialyzed thoroughly against storage buffer (20 mM Tris 8.0, 150 mM NaCI, 2 mM TCEP, 5% glycerol) using 10K MWCO dialysis cassettes (Pierce Slide-a-lyzer, Biotechnology Inc., Rockford, IL) to remove imadizole. Ni-NTA purified protein yield is then estimated by A280 (1 A280 = 0.77 mg / mL). Ni-NTA purified protein is cleaved with Thrombin (High Activity Thrombin - Calbiochem) at 5 Units / mg. Cleaved protein is then repurified over Ni-NTA resin (adding 20 mM Imadizole to the sample before loading) using same Ni-NTA buffers. Flow- thru containing cleaved protein is collected and concentrated to approx 10 mg / mL (using 10K MWCO concentrator). Concentrated protein is then purified over SX200 column in crystallization buffer (20 mM Tris 7.3, 150 mM NaCI, 2 mM TCEP, 5% glycerol). SX200 purified protein is stored on ice until used for crystallization trials. Example 3: Crystallization of PDE10A wild-type catalytic domain with compound of Formula 1 [2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)- phenoxy]-acetic acid)

Compound of Formula I in 100% DMSO at 3OmM was added to SX200 pure protein at to a final concentration of 300 uM and incubated on ice for 30 min. Protein complex was then concentrated (using 10K MWCO concentrator) to 8mg / mL for crystallization trials. Concentration was assessed by Protein Determination using Coomassie Plus Protein Reagent (Pierce, Biotechnology Inc., Rockford, IL). PDE9 co-crystals were grown using the vapor diffusion method in 24 well VDX plates (Hampton Research, Aliso Viejo, CA). Crystals were grown in 19 - 21% Polyacrylic Acid, 0.1 M MES pH 6.5, 0.2M MgCI2 (magnesium chloride) using 1 :1 drop ratios of protein : precipitant. Drop size was 3 uL and precipitant well volume was 800 uL. Plates were incubated at 220C.

Example 4: X-ray data collection, structure determination and refinement of PDE9A: compound of Formula 1 ([2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3- d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid) complex

The crystals prepared in Example 3 were transferred to a cryoprotectant solution, made up of the reservoir solution, with 30% glycerol, and then flash-frozen in a stream of cold nitrogen gas at 100K or in liquid nitrogen. A full data set was collected from one crystal frozen in this manner at synchrotron beam line COMCAT at APS in Chicago. Data were processed using the HKL2000 suite of software (Otwinowski, Z. & Minor, W. Methods Enzymology 276, 307-326 (1997). Data collection statistics are summarized in Table 5a. The crystals belong to space group I422 with unit cell dimensions a=b=223.665, c=119.341 A, α=β=γ=90.0°. They contain 2 molecule of the polypeptide, and two molecule of the inhibitor per asymmetric unit. The structure was solved by the method of molecular replacement, using the program MOLREP (Vagin A. & Teplyakov, A. Acta Crystl D56, 1622 (2000)). A homology model of PDE9, based on the previously determined structure of PDE 10 was used as the search model. A clear solution to the rotation/translation search was found, with a starting R-factor of 47.8% for data to 2.5A. The final model was built with manual rebuilding on the graphics screen, using the program O. Refinement in Refmac was carried out using all data in the resolution range 30.0-2.7A. Partial structure factors from a bulk-solvent model and anisotropic B-factor correction were supplied throughout the refinement. The R-factor for the current model is 0.22 (free R-factor, 5% of the data, 0.27). The refinement statistics are summarized in Table 5b. The current model contains 316 in molecule A out of 323 amino acid residues calculated on the basis of the construct, and 308 in molecule B. lnterpretable electron density is seen for all residues from 242-557 in molecule A and 250-557 in molecule B. Residues 239- 241 in molecule A, and residues 239-249 in molecule B, at the N-terminus and 558-562 in both molecules have not been modelled. In addition, the model contains one Zn2+ ion, one Mg2+ ion, one molecule of the inhibitor, the compound of Formula 1 ([2-(3-lsopropyl-7-oxo-6,7- dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)-phenoxy]-ace tic acid), for each protein molecule, and 49 water molecules. A schematic representation of [2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3- d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid is given below:

Formula I

[2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)-phenoxy]- acetic acid

The compound of Formula 1 is disclosed in pending United States Patent Application filed on April 20, 2004 entitled "PDE9 INHIBITORS FOR TREATING TYPE 2 DIABETES, METABOLIC SYNDROME, AND CARDIOVASCULAR DISEASE" and is hereby expressly incorporated by reference in its entirety. Table 5a -Data statistics

1 Numbers in parentheses refer to the highest resolution range (2.7-2.8A) 2 Rsym = Σ(l-<l>)/Σ <l>

Table 5b- Refinement statistics

3 D R - = Σ||Fobs| - /f|Fcalc||/Σ|Fobs|

Equivalents While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims should be interpreted by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.